DESCRIPTION: This proposal is designed to study properties of glutathione (GSH) S-transferases (GSTs) and mechanisms of regulation of expression of their genes. GSTs are abundant proteins that function both as enzymes and as stoichiometric binding proteins. GSTs use GSH to catalyze biotransformations of electrophilic compounds and reduction of organic hydroperoxides. GSTs also function as intracellular high-capacity binding proteins for a wide variety of non-polar compounds including hormones and drugs. In some cases GSTs can counteract the cytotoxic or genotoxic effects of such agents. Dietary components or substances from the environment that elevate levels of GSTs and other Phase II drug metabolizing enzymes are thus thought to be chemoprotective or anticarcinogenic agents. However, very little is known about mechanisms by which expression of human GST genes are regulated. The applicant has recently identified cis-acting regulatory sequences in the 5-flanking regions of human mu-class GST genes. These include a tandem ARE-XRE motif in the case of the GSTM4 gene and multiple NFk-B motifs for the GSTM3 gene. To define the DNA sequences required for basal or uninduced transcription and to uncover changes after exposure of the cells to certain hormones, xenobiotics, and growth factors, a series of constructs spanning the promoter regions of these genes and including specific deletions and mutations, will be cloned into expression plasmids that contain the luciferase reporter gene. These reporter vectors will be transfected into human cells. The use of human genes expressed in human cell lines is intended to facilitate elucidation of relationships of levels of transcriptional enhancement obtained with reporter constructs to effects on protein products of the genes. The proteins will be resolved by HPLC and identified using specific antisera. Structures of different classes of GSTs have recently been solved by X-ray crystallography, structural data will be exploited to probe ligand binding mechanisms for individual GSTs by affinity-labeling and spectroscopic methods. Since GSTs are products of gene superfamilies, study of their functions in cells is complex, if traditional methods such as antisense technology or homologous recombination in embryonic stem cells are used. The study of subunit assembly mechanisms is emphasized in order to design methods to control GST activity at the level of the protein. Using recombinant DNA - methods and site-directed mutagenesis it is proposed that the applicant will prepare GST subunits defective in and interfering with GST functions but retaining their capacity to assemble as dimers. The genes for the chimeric subunits will be introduced into cells and overexpressed; in the cells the chimeric subunits are considered likely to recombine with an inactivate an entire class of corresponding natural subunits. Effects of GST depletion at the protein level well then be studied.